The alveolar epithelium is directly exposed to variations in alveolar O2 tension in response to many physiological or pathological conditions. For example, alveolar hypoxia may be the consequence of obstructive airway diseases, or pulmonary edema from heart failure or acute lung injury. Although the alveolar epithelium is directly exposed during hypoxia, limited information has been obtained about the effects of low O2 tension on alveolar epithelial cell functions. In the previous cycle of this proposal, we reported that hypoxia causes significant remodeling of the alveolar epithelial cytoskeleton. In particular, we observed that hypoxia initiates rapid and localized restructuring of the keratin intermediate filament network and interacted with multiple signal transduction pathways which permits the keratin network to be involved in an intimate crosstalk with many aspects of cell behavior including migration, proliferation, and apoptosis. This proposal will develop the hypothesis that keratin intermediate filaments can dynamically respond to low O2 tension by initiating a cytoprotective reorganization of the network that is mediated by changes in the phosphorylation state of keratin 8 and 18. We have formulated three interrelated specific aims to study the hypoxia-induced regulation of keratin IFs in the alveolar epithelium. Specific Aim #1. To identify the in vivo phosphorylation sites in keratin 8 and 18 involved in the hypoxia-mediated restructuring of the keratin intermediate filament network in alveolar epithelial cells. Specific Aim #2. To determine whether reconstituting wild-type or phospho-mutant keratin proteins in alveolar epithelial cells and in keratin knockout mice restores alveolar epithelial function. Specific Aim #3. To determine whether hypoxia inducible factor is required for the hypoxia-mediated transcriptional regulation of keratin 8 and/or keratin 18 genes in alveolar epithelial cells. The purpose of this proposal is to test the hypothesis that hypoxia regulates the state of phosphorylation of K8 and K18 proteins, which mediates the assembly dynamics and micromechanical properties of the KIF network. The KIF network plays an important contributory role to the cellular integrity of alveolar epithelial cells. The proposed experiments will determine the molecular mechanisms that regulate the hypoxia mediated reorganization and/or disassembly of keratin IFs. The consequences of this reorganization on alveolar epithelial function will be examined both in vitro and in vivo using primary ATII cells, wild-type and keratin-deficient mice. Completion of the proposed studies will provide novel insights on the role of keratin IF in the pathogenesis of hypoxia-induced alveolar epithelial dysfunction, which is of biological and physiological importance in patients with pulmonary edema.